Colorimetric ELISA with an acid–base indicator for sensitive detection of ochratoxin A in corn samples

Abstract

In this study, a direct competitive enzyme-linked immunosorbent assay (dcELISA) with an acid–base indicator was developed for the naked-eye detection of ochratoxin A (OTA). OTA-labeled urease conjugates were used as competing antigens, and the acid–base indicator of bromocresol purple (BCP) dye was employed as the signal reporter. When the competing antigen was captured by the anti-OTA antibody on the plate well, the urea substrate was catalyzed to generate NH₃ molecules. The BCP dye produced a vivid color change from purple to light yellow because of a slight pH change in the solution, and hence, the indicator was suitable for the naked-eye detection of analytes. Under optimized conditions, the proposed method exhibited a high sensitivity for OTA naked-eye detection with a cutoff limit of 50 pg mL⁻¹. The method also showed a good linear range of 12.5–100 pg mL⁻¹ for OTA quantitative detection, with a half maximal inhibitory concentration of 31.4 pg mL⁻¹, which is 11-fold lower than that of conventional HRP-based ELISA (IC₅₀ = 354 pg mL⁻¹). The reliability of the proposed colorimetric ELISA method for naked-eye detection indicated no false negative and only three false positive results from 70 tests. Intra- and inter-assays showed that the average recovery ranged from 83.7% to 111%, and the coefficient of variation (CV) ranged from 3.71% to 13.5%. The proposed method exhibited acceptable accuracy and precision for the quantitative detection of OTA in real corn samples. In summary, the developed acid–base indicator-based dcELISA offers a promising platform for resource-constrained countries for highly sensitive naked-eye detection of haptens.